The flavonoids are a major class of phenylpropanoid-derived plant natural products. Their fifteen carbon (C6–C3–C6) backbone can be arranged as a 1,3-diphenylpropane skeleton (flavonoid nucleus) or as a 1,2-diphenylpropane skeleton (isoflavonoid nucleus). Although 1,3-diphenylpropane flavonoid derivatives are almost ubiquitous among terrestrial plants, the 1,2-diphenylpropane isoflavonoids are restricted primarily to the Leguminosae, although they occur rarely in other families such as the Apocynaceae, Pinaceae, Compositae, and Moraceae (Tahara, S. and R. K. Ibrahim, 1995, “Prenylated isoflavonoids—an update,” Phytochemistry 38: 1073–1094).
The limited taxonomic distribution of the isoflavonoids is directly related to the occurrence of the enzyme complex isoflavone synthase (IFS), which catalyzes the aryl migration reaction leading to the formation of an isoflavone from a flavanone. While flavanones are ubiquitous in higher plants, the IFS reaction, which is a two-step process specific for isoflavonoid biosynthesis (Kochs, G. and H. Grisebach, 1986, “Enzymic synthesis of isoflavones,” European J Biochem 155: 311–318), is limited to the Leguminosae and the other diverse taxa in which isoflavonoids are occasionally found.
The presence of isoflavonoids provides several advantages to plants. One such advantage is provided by the function of isoflavonoids as antimicrobial phytoalexins in plant-microbe interactions. For example, the simple isoflavones daidzein and genistein act as initial precursors in the biosynthesis of various antimicrobial isoflavonoid phytoalexins in a wide variety of legumes (Dixon, R. A. and N. L. Paiva, 1995, “Stress-induced phenylpropanoid metabolism,” Plant Cell 7: 1085–1097). Isoflavonoid compounds have been shown to accumulate in infected plant cells to levels known to be antimicrobial in vitro. The temporal, spatial and quantitative aspects of accumulation are consistent with a role for these compounds in disease resistance (Rahe, J. E., 1973, “Occurrence and levels of the phytoalexin phaseollin in relation to delimitation at sites of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum,” Canadian J Botany 51: 2423–2430; Hadwiger, L. A. and D. M. Webster, 1984, “Phytoalexin production in five cultivars of pea differentially resistant to three races of Pseudomonas syringae pv. pisi,” Phytopathology 74: 1312–1314; Long, et al., 1985, “Further studies on the relationship between glyceollin accumulation and the resistance of soybean leaves to Pseudomonas syringae pv. glycinea,” Phytopathology 75: 235–239; Bhattacharyya, M. K. and E. W. B. Ward, 1987, “Biosynthesis and metabolism of glyceollin I in soybean hypocotyls following wounding or inoculation with Phylophihora megasperma f. sp. glycinea,” Physiol and Mol Plant Pathology 31: 387–405). Moreover, it has been reported that many plant pathogens are much more sensitive to phytoalexins of non-host species than they are to the phytoalexins of their natural hosts, because they can often detoxify the host's phytoalexins. (VanEtten, et al., 1989, “Phytoalexin detoxification: importance for pathogenicity and practical implications,” An Rev Phytopathology 27: 143–164).
Isoflavonoids also function in plant-microbe interactions in the establishment of bacterial or fungal symbioses with plants. Isoflavonoids have been reported to regulate bacterial nodulation genes, acting as a major nod gene inducer (Kosslak, et al., 1987, “Induction of Bradyrhizobium japonicum common nod genes by isoflavones isolated from Glycine max,” Proc Natl Acad Sci USA 84: 7428–7432) and/or transcription activator (Dakora, et al., 1993, “Common bean root exudates contain elevated levels of daidzein and coumestrol in response to Rhizobium inoculation,” Mol Plant-Microbe Interact 6: 665–668). Isoflavonoids have also been shown to have a role on the establishment of the symbiotic vesicular arbuscular mycorrhizal (VAM) association of the fungus Glomus with legume roots. (Kape, et al., 1992, “Legume root metabolites and VA-mycorrhiza development,” J Plant Physiol 141: 54–60). Xie et al have reported that the isoflavonoids coumestrol, daidzein and genistein have small but significant stimulatory effects on the degree of mycorrhizal colonization of soybean, and that one effect of isoflavonoids on the soybean mycorrhizal symbiosis could be via induction of nodulation factors from co-colonizing Rhizobia, since nod-factors have also been shown to stimulate fungal colonization (Xie, et al., 1995, “Rhizobial nodulation factors stimulate mycorrhizal colonization of nodulating and nonnodulating soybeans,” Plant Physiology 108: 1519–1525).
In addition to the advantages that the presence of isoflavonoids confers to plants, a significant body of evidence indicates that dietary consumption of isoflavonoids can provide benefits to human health. Dietary isoflavones have been ascribed strong cancer chemopreventative activity in humans, and display a range of pharmacological activities suggestive of various other health promoting effects, including phytoestrogen activity as both estrogenic and anti-estrogenic agents (Coward, et al., 1993, “Genistein, daidzein, and their -glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets,” J Agricultural and Food Chemistry 41: 1961–1967; Martin, et al., 1996, “Interactions between phytoestrogens and human sex steroid binding protein,” Life Sciences 58: 429–436); anticancer effects associated with phytoestrogenic activity (Lee, et al., 1991, “Dietary effects on breast-cancer risk in Singapore,” Lancet 337: 1197–1200; Adlercreutz, et al., 1991, “Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet,” Am J Clin Nutr 54: 1093–1100); anticancer effects associated with inhibition of several enzymes including DNA topoisomerase and tyrosine protein kinase (Akiyama, et al., 1987, “Genistein, a specific inhibitor of tyrosine-specific protein kinases,” J Biol Chem 262: 5592–559; Uckun, et al., 1995, “Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases,” Science 267: 886–891); suppression of alcohol consumption (Keung, W. M. and B. L. Vallee, 1993, “Daidzin: A potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase,” Proc Natl Acad Sci USA 90: 1247–1251; Keung, et al., 1995, “Daidzin suppresses ethanol consumption by Syrian golden hamsters without blocking acetaldehyde metabolism,” Proc Natl Acad Sci USA 92: 8990–8993); antioxidant activity (Arora, et al., 1998, “Antioxidant activities of isoflavones and their biological metabolites in a lipsomal system,” Arch Biochem Biophys 356: 133–141; Tikkanen, et al., 1998, “Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance,” Proc Natl Acad Sci USA 95: 3106–3110); effects on calcium metabolism, some of which may be linked to protective effects against osteoporosis (Tomonaga, et al., 1992, “Isoflavonoids, genistein, PSI-tectorigenin, and orobol, increase cytoplasmic free calcium in isolated rat hepatocytes,” Biochem Biophys Res Com 182: 894–899; Draper, et al., 1997, “Phytoestrogens reduce bone loss and bone resorption in oophorectomized rats,” J Nutr 127: 1795–1799); and cardiovascular effects (Wagner, et al., 1997, “Dietary soy protein and estrogen replacement therapy improve cardiovascular risk factors and decrease aortic cholesteryl ester content in ovariectomized cynomolgus monkeys,” Metabolism—Clinical and Experimental 46: 698–705).
At present, the only dietary sources of isoflavonoids for humans are certain legumes such as soybean or chickpea. The development of methods to genetically manipulate isoflavonoids in plants, either to widen the source of dietary isoflavonoids for humans, or to exploit the biological activities of isoflavonoids for plant protection and improvement, is wholly dependent on the availability of cloned genes encoding the various enzymes of isoflavonoid biosynthesis. Of these, the isoflavone synthase (IFS) complex constitutes the first committed reactions, and as such represents the means to introduce isoflavonoids into plants that do not possess the pathway.
In 1984, Hagmann and Grisebach provided the first evidence for the enzymatic conversion of flavanone to isoflavone (the IFS reaction) in a cell free system (Hagmann, M. and H. Grisebach, 1984, “Enzymatic rearrangement of flavanone to isoflavone,” FEBS Letters 175: 199–202). They demonstrated that microsomes from elicitor-treated soybean cell suspension cultures could catalyze the conversion of 2(S)-naringenin to genistein, or of 2(S)-liquiritigenin to daidzein, in the presence of NADPH. The crude microsomal enzyme preparation, which was stable at −70° C. but had a half-life of only 10 minutes at room temperature, was absolutely dependent on NADPH and molecular oxygen. It was subsequently shown that the reaction proceeded in two steps. The flavanone was converted in a cytochrome P450-catalyzed reaction requiring NADPH and O2 to the corresponding 2-hydroxyisoflavanone. This relatively unstable compound, which could, however, be identified by mass spectrometric analysis, then underwent dehydration to yield the isoflavone. The dehydration reaction appeared to be catalyzed by an enzyme present predominantly in the cytoplasmic supernatant, although it was not possible to remove all this activity from the microsomes. The corresponding 2-hydroxyisoflavanone spontaneously converted to genistein, for example, in methanol at room temperature. Kinetic analysis indicated that the 2-hydroxyisoflavanone was formed prior to genistein, consistent with its being an intermediate in isoflavone formation. (Kochs, G. and H. Grisebach, 1986, “Enzymic synthesis of isoflavones,” European J Biochem 155: 311–318).
Involvement of cytochrome P450 in the 2-hydroxyisoflavanone synthase reaction was confirmed by inhibition by CO, replacing O2 with N2, and examining the effects of a range of known P450 inhibitors of which ancymidol was the most effective. The enzyme co-migrated with the endoplasmic reticulum markers cinnamate 4-hydroxylase (another cytochrome P450) and cytochrome b5 reductase on Percoll gradients. The enzyme is stereoselective, and (2R)-naringenin is not a substrate. (Kochs, G. and H. Grisebach, 1986, “Enzymic synthesis of isoflavones,” European J Biochem 155: 311–318).
The origin of the 2-hydroxyl group was determined from studies on the IFS present in microsomes from elicited cell cultures of Pueraria lobata. 18O from 18O2 was incorporated into the 2-hydroxyl group, resulting in a 2-hydroxyisoflavanone with molecular ion shifted by two mass units, whereas there was no corresponding shift in the molecular ion of daidzein, consistent with the subsequent dehydration reaction (Hashim, et al., 1990, “Reaction mechanism of oxidative rearrangement of flavanone in isoflavone biosynthesis,” FEBS Letters 271: 219–222). The currently accepted model for the reaction pathway of IFS as illustrated in FIG. 1, therefore, involves P450-catalyzed hydroxylation coupled to aryl migration, a reaction with mechanistic similarities to the well described proton migration mechanism of some P450 reactions (Hakamatsuka, et al., 1991, “P450-dependent oxidative rearrangement in isoflavone biosynthesis: reconstitution of P-450 and NADPH:P450 reductase,” Tetrahedron 47: 5969–5978).
Currently, there have been no reports on purification to homogeneity or molecular cloning of the cytochrome P450 of the IFS complex because of the extreme lability of the enzyme. The 2-hydroxyisoflavanone synthase cytochrome P450 from Pueraria has been solubilized with Triton X-100, and partially purified by DEAE-Sepharose chromatography; the enzymatic reaction could be reconstituted by addition of NADPH cytochrome P450 reductase that separated from the hydroxylase on the ion exchange column (Hakamatsuka, et al., 1991, Tetrahedron 47: 5969–5978). A 2-hydroxyisoflavanone dehydratase has been purified from elicitor-treated P. lobata cells, and has been shown to be a soluble monomeric enzyme of subunit Mr 38,000 (Hakamatsuka, et al., 1998, “Purification of 2-hydroxyisoflavanone dehydratase from the cell cultures of Pueraria lobata,” Phytochemistry 49: 497–505). It is not yet clear whether this enzyme physically associates with the P450 hydroxylase catalyzing the aryl migration, or even whether this activity is essential for isoflavone formation in planta in view of the spontaneous conversion of 2-hydroxyisoflavanone to isoflavone.
Flavanone is a potential substrate for more than one type of hydroxylation reaction at the 2-position. Thus, elicitor-treated cell cultures of alfalfa and Glycyrrhiza echinata have been shown to accumulate the dibenzoylmethane licodione (Kirikae, et al., 1993, “Biosynthesis of a dibenzoylmethane, licodione, in cultured alfalfa cells induced by yeast extract,” Biosci Biotech Biochem 57: 1353–1354). Licodione synthase is, by classical criteria, a cytochrome P450, the activity of which is induced by yeast elicitor in Glycyrrhiza cells (Otani, et al., 1994, “Licodione synthase, a cytochrome P450 monooxygenase catalyzing 2-hydroxylation of 5-deoxyflavanone, in cultured Glycyrrhiza echinata L. cells,” Plant Physiol 105: 1427–1432). The reaction it catalyzes involves 2-hydroxylation of flavanone followed by hemiacetal opening instead of aryl migration, and the reaction was thought to have mechanistic similarities to the flavone synthase II enzyme previously characterized from soybean (Kochs, G. and H. Grisebach, 1987, “Induction and characterization of a NADPH-dependent flavone synthase from cell cultures of soybean,” Z. Naturforsch 42C: 343–348). A gene encoding the flavone synthase II/licodione synthase from Glycyrrhiza has been cloned (Akashi, et al., 1998, “Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase II,” FEBS Letters 431: 287–290), and a different cytochrome P450 gene encoding flavone synthase II has recently been cloned from Gerbera hybrida (Martens, S. and G. Forkmann, “Cloning and expression of flavone synthase II from Gerbera hybrids,” Plant J 20: 611–618).
Although the reactions catalyzed by IFS are critical for the formation of all isoflavonoids in plants, there have been no previous reports of the isolation of genes encoding components of isoflavone synthase, although genes encoding most of the other enzymes of the isoflavonoid pathway, including downstream enzymes converting simple isoflavones to antimicrobial phytoalexins, have been characterized (Dixon, et al., 1995, “The isoflavonoid phytoalexin pathway: from enzymes to genes to transcription factors,” Physiologia Plantarum 93: 385–392). Thus, the unavailability of isoflavone synthase genes has made it heretofore impossible to utilize the downstream genes for regulating isoflavonoid concentrations in legumes and other plants that do have the isoflavonoid pathway, or for engineering antimicrobial and pharmacologically active isoflavonoids in transgenic plants of species that do not have the isoflavonoid pathway.
Genes encoding the enzyme catalyzing the first step of the isoflavone synthase reaction have now been isolated and purified from soybean and Medicago truncatula (barrel medic).